Ubiquitin is a 76-residue protein that exists in cells either free or conjugated to many other proteins. Selective degradation of proteins by the Ub/proteasome system plays a central role in a multitude of biological processes, including cell growth, differentiation, and responses to stress. The work of this laboratory focuses, at present, on a universally present pathway of the ubiquitin system called the N- end rule pathway. The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. The objective of the research described in this renewal application is to advance the understanding of the ubiquitin system through biochemical and genetic dissection of the mammalian (mouse) N-end rule pathway. The other NIH grant(DK39520) that supports research by this laboratory focuses on the yeast (S. cerevisiae) N-end rule pathway. Specific Aims 1) Biochemical and genetic dissection of the mouse Asn- specific N-terminal amidase NTAN1p, a component of the N-end rule pathway; studies of the recently constructed mouse strains that lack Ntan1. 2) Biochemical and genetic dissection of the mouse Arg-tRNA- protein transferase ATE1p, a component of the N-end rule pathway; analysis of the recently cloned mouse Ate1 gene and its product; construction and study of mouse strains that lack Ate1. 3) Biochemical and genetic dissection of the mouse N-recognin UBR1p, the E3 enzyme of the mammalian N-end rule pathway, and of its recently identified homologs UBR2p and UBR3p; investigation of the recently constructed mouse strains that lack Ubr1; construction and study of mouse strains that lack Ubr2 and/or Ubr3. 4) Definition and analysis of ubiquitin- dependent degradation signals in mouse and Xenopus c-Mos, a conditionally short-lived Ser/Thr-kinase and a proto-oncoprotein. 5) A screen for physiological substrates of the mammalian N-end rule pathway that utilizes sib selection strategy in reticulocyte extracts. 6) The N-end rule pathway in the nucleus: proof of existence and further analysis.